Nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to attempts to develop them for early diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR), ligase chain reaction (LCR) and transcription-based amplification require repeated cycling of the reaction between high (85.degree. C.-100.degree. C.) and low (30.degree. C.-40.degree. C.) temperatures to regenerate single stranded target molecules for amplification. In contrast, methods such as Strand Displacement Amplification (SDA), self-sustained sequence replication (3SR) and the Q.beta. replicase system are isothermal reactions which can be performed at a constant temperature. Conventional SDA (performed at lower temperatures, usually about 35.degree.-45.degree. C.) is described by G. T. Walker, et al. (1992a. Proc. Natl. Acad. Sci. USA 89, 392-396 and 1992b. Nuc. Acids. Res. 20, 1691-1696). A thermophilic version of the SDA reaction (tSDA, described below) has recently been developed, and is performed at a higher, but still constant, temperature using thermostable polymerases and restriction endonucleases. Thermophilic SDA is performed essentially as conventional SDA, with substitution of a thermostable polymerase and a thermostable restriction endonuclease. The temperature of the reaction is adjusted to a higher temperature suitable for the selected thermophilic enzymes and the conventional restriction endonuclease recognition/cleavage site is replaced by the appropriate restriction endonuclease recognition/cleavage site for the selected thermostable endonuclease.
Nucleic acid amplification reactions are very reproducible and highly efficient in "clean" systems where the target sequence to be amplified is in a compatible buffer and is essentially free of non-nucleic acid molecules. In a clinical setting, however, the target sequence is generally in a biological sample which also contains a wide variety of non-nucleic acid molecules (e.g., proteins, carbohydrates, lipids, etc.). Nucleic acid amplification in biological samples has often been inconsistent and variable, presumably because the other biological molecules which are present interfere partially or totally with the amplification reaction. This leads to false negative results and an inability to accurately quantify the amount of target sequence present. Further, a strongly positive sample may appear to be a weak positive in a sample where amplification is inhibited. These problems may make it difficult to accurately diagnose and effectively treat a patient based on the results of a test based on nucleic acid amplification.
Little is known about specific inhibitory molecules or the mechanisms of inhibition, however. For this reason prior art methods for reducing or eliminating inhibition of nucleic acid amplification in biological samples have been relatively non-specific and have been directed to the general removal or degradation of proteins in the sample. For example, phenol extraction of the biological sample has been used as a general method for removing hydrophobic proteins. Hydroxylated surfaces have been used to bind proteins for separation from the sample, and protease treatment has been used to non-specifically degrade proteins. In many cases, it is also possible to overcome the effect of inhibitors by diluting the sample prior to amplification. However, this approach also dilutes the target being amplified and thereby reduces the sensitivity of the assay. An alternative approach has been to isolate the nucleic acid from non-nucleic acid molecules, e.g., by binding to silica. While isolation of the nucleic acids may often successfully eliminate inhibitors of amplification, the process is time-consuming and recovery of the target is generally nonquantitative so the sensitivity of the assay is compromised. The success of such general methods has been variable, which may be at least partially attributable to varying amounts and types of inhibitory molecules in different biological samples.
It has been particularly difficult to obtain consistent nucleic acid amplification in sputum samples, as this type of biological sample is often especially inhibitory in the amplification reaction. Sputum is, however, a very important specimen for the diagnosis of pulmonary diseases such as tuberculosis. As they are generally very viscous (especially when pulmonary disease is present) sputum samples are typically liquified prior to analysis. A commonly used sample processing method for analysis of Mycobacteria in sputum is the N-acetyl-L-cysteine/sodium hydroxide method. This method uses NaOH, sodium citrate and N-acetyl L-cysteine (NALC) to liquify the sample, with recovery of the mycobacteria by centrifugation. The pellet is then resuspended in a small volume and used for culture or other diagnostic tests. Similar methods have been developed in which sodium hydroxide is used alone, with neutralization of the pellet prior to resuspension and analysis. NaOH has also been used with sodium lauryl sulfate (SLS) to process sputum samples, again with neutralization of the pellet by addition of acid prior to culture or other testing. In most cases, amplification inhibitors are found in the pellet after the centrifugation step, along with the Mycobacteria to be analyzed. The supernatant of a significant number of samples may also be inhibitory. These are the samples which present the greatest difficulty for nucleic acid amplification, as the results are inconsistent, variable and difficult to interpret.
It has now been discovered that certain glycoproteins, and mucins in particular, are significant inhibitors of nucleic acid amplification. Glycoproteins are proteins which contain covalently linked carbohydrate moieties. They are particularly prevalent in mammalian tissues. The carbohydrate chains of glycoproteins are mainly comprised of seven sugars: D-galactose, N-acetyl-D-galactosamine (GalNAc), D-glucose, N-acetyl-D-glucosamine, D-mannose, L-fucose, and sialic acid (N-acetylneuraminic acid, NGNA). The mucins are a type of glycoprotein with a complex carbohydrate structure containing disaccharide groups of N-acetylneuraminyl-(2.fwdarw.6)-N-acetylgalactosamine. Mucopolysaccharides (polysaccharides comprised of amino sugars or their derivatives) are also often found covalently bound to protein. Although the amino sugars of many mucopolysaccharides are the same as those found in glycoproteins, they are generally considered as a separate group of molecules because mucopolysaccharide-protein structures are much richer in carbohydrate than most glycoproteins. In the case of sputum samples, which often contain high concentrations of mucins, conventional sample processing methods such as liquification appear to increase the inhibitory effect of glycoproteins. The discovery of these mechanisms of amplification inhibition has led to the development of methods targeting glycoproteins to reduce their inhibitory effect.